Vaginal composition comprising a combination of estrogen and vitamin D

ABSTRACT

The invention relates to vaginal composition comprising a combination of estrogen and vitamin D or a vitamin D analog at a daily dosage delivery of (i) estrogen ranging from 1 μg to 100 μg estrogen of estradiol equivalent and (ii) vitamin D or analog ranging from 7.5 μg to 100 μg of vitamin D equivalent.

FIELD OF THE INVENTION

The present invention relates to the field of treating vaginal atrophy.

BACKGROUND OF THE INVENTION

Women experienced many physical changes during the period of menopausetransition and after menopause including vasomotor symptoms (hotflashes, night sweats), sleep disturbance, mood changes and alsovulvovaginal symptoms, urinary incontinence and osteoporosis. All ofthese symptoms or related health conditions are caused by a decrease inestrogens and other hormones and the effects of aging.

Vulvovaginal symptoms include vaginal dryness, vulvovaginalirritation/itching, and dyspareunia, and are experienced by an estimatedfrom about 4% in the early premenopausal groups to 47% in the latepostmenopausal group (Mc Bride M et al., 2010 “Vulvovaginal atrophy”.Mayo Clin Proc; 85 (1):87-94). In contrast to vasomotor symptoms, whichusually abate over time even without treatment; symptomatic vaginalatrophy is typically progressive and unlikely to resolve on its own(NAMS, 2010,“Contemporary Clinical Management of Menopause”).Vulvovaginal atrophy (VVA) is characterized by different physical signs.The mucosa of the cervix, the epithelium of the vagina and vulva thinand become susceptible to injury; the vaginal rugae diminish leading toa smoother appearing vaginal wall which is accompanied by diminishedblood flow (Sturdee D W et al., 2010. Climacteric: 1-24). Leftuntreated, severe vulvovaginal atrophy can result in a vaginal surfacethat is friable, with petechial, ulcerations and tears, accompanied insome cases by stenosis. Bleeding may occur from minimal trauma: wiping,speculum insertion and also during intercourse (NAMS, 2013, “Managementof symptomatic vulvovaginal atrophy: 2013 Position statement of theNorth American Menopause Society”. Menopause, 20;(9):888). Consequentlybefore irreversible changes occur, early detection and treatment ofvaginal atrophy should be implemented. Moreover long-term therapy may benecessary to maintain vaginal (urogenital) health.

One of the options for postmenopausal women with symptoms and quality oflife issues is to receive hormone replacement therapy (estrogen alone ora combination of estrogens and progestins). While systemic hormonereplacement therapy is effective in abating vasomotor-relateddiscomfort, 25% to 40% of women using systemic treatment stillexperience persistent vaginal dryness (Johnston S L. Farrell S A.Bouchard C et al. The detection and management of vaginal atrophy. J.Obstet. Gynaecol. Can. 26, 503-515 (2004)). Moreover, the systematicadministration of unopposed estrogen has been shown to be associatedwith an increased risk for endometrial hyperplasia and carcinoma in adose- and duration-dependent manner in postmenopausal women who have notgone hysterectomy (Furness S et al., “Hormone therapy in postmenopausalwomen and risk of endometrial hyperplasia”. Cochrane database Syst Rev2009/CD000402). Thus, if vasomotor symptom relief or osteoporosisprevention is not indicated, recommended treatment for womenexperiencing vulvovaginal atrophy is local estrogen application withdrugs providing minimal systemic absorption.

Vaginal estrogen products deliver estrogen locally to vaginal tissueswith systemic absorption proportional to dose used. Various vaginalestrogen preparations such as conjugated equine estrogens (Premarin®),estradiol (Estrace®) vaginal creams, a sustained-release intra-vaginalestradiol ring (Estring®) and a low-dose estradiol (Vagifem®) andestriol tablets are useful therapeutic options in the treatment of thiscondition. All treatments provided equivalent relief of the symptoms ofVVA based on composite scores of vaginal symptoms (dryness, soreness,and irritation) (Suckling J. et al., 2006, Cochrane Database Syst Rev.Oct 18;(4):CD00150). Although intravaginal formulations were developedto avoid systemic exposure to estrogens, several studies havedemonstrated that all vaginal products lead to significant increases inscrum estrogen levels and thus that systemic action is expected (LabrieF et al., 2009, Menopause. January-February; 16(1):30-6).

Safety information, especially regarding long-term exposure associatedwith local estrogen therapy is not well documented in the literature tosupport an assumed more favorable risk-benefit ratio for endometrialhyperplasia and carcinoma. Even if it is rare, endometrial hyperplasiahas been seen with low-dose vaginal estrogens (Bachmann G. et al., 2008,Obstet Gynecol. January; 111(l):67-76. Suckling J. et al., 2006.Cochrane Database Syst Rev. Oct 18;(4):CD00150). Such risk is notacceptable for patients.

Ultra-low dose of estrogen have been evaluated with the aim to avoidsystemic effects and to reduce side effects. For instance, the 10 μgestradiol vaginal tablets Vagifem has been developed to offer the lowesteffective hormonal dose (Panay N et al., 2012. Menopause: 18(1):15-9).Even at these dosages, a minor degree of systemic absorption may occurin some patients, especially during the first two weeks of treatment. Inan open-label, multicenter trial on 386 women, incidence rate ofhyperplasia and/or carcinoma was 0.52% (95% CI 0.06%, 1.86%) at the endof 52 weeks treatment. Improvements on vulvovaginal atrophy aresignificantly higher than for the placebo but as expected lower thanwith increased doses (the 25 meg estradiol vaginal tablet for instance)(Chollet J A., 2011, Patient preference and adherence:5:571-574).

Overall, the improvement of symptoms with local estrogen therapy iscurrently not optimal: as an example, improvement of objective symptomswith Vagifem 25 mcg/day vs placebo has been only 41.4% (Chollet J A.,2011, Patient preference and adherence; 5:571-574).

Several authors have evaluated alternative treatments to avoid estrogenuse, especially to overcome potential risk of endometrial hyperplasiaand carcinoma. Conflicting reports suggest the use of vitamin D would bebeneficial on vaginal atrophy. Yildirim has shown that oral vitamin Dsupplementation (0,500 meg calcitriol/day) for at least one year inpostmenopausal women resulted in squamous maturation of the vaginalepithelium compared to non-supplemented postmenopausal women (Yildirim Bet al., 2004. Maturitas Dec 10:49(4):334-7). Similarly, Rad tested theeffect of vaginal vitamin D (suppository 1000 IU vit D Rocatrol, dailyfor 8 weeks) on vaginal atrophy in 44 postmenopausal women anddemonstrated a significant increase of superficial cells in the vaginalepithelium and a significant decrease in vaginal pH. (Rad P et al.,2015, Iran J Nurs Midwifery Res., March-April; 20(2):211-5). On thecontrary, results from the Women Health Initiative trial suggest thatsupplementation with 1000 mg of calcium and 400 IU of vitamin D does notinfluence menopause-related symptoms, including vaginal dryness, over anaverage of 5.7 years of follow-up among postmenopausal women (Leblanc Eet al., Maturitas. 2015 Jul; 81(3):377-83). In osteoporotic womendiscontinuing estrogen replacement therapy, Checa came to a similarconclusion and demonstrated a significant decrease in vaginal maturationvalue in the oral calcium (500 mg/day) plus vitamin D3 (400 IU/day)group one after one year of treatment (Checa MA et al., Maturitas. 2005September 16; 52(1):70-7.) Thus there is no consensus on the effect ofvitamin D on vaginal atrophy.

Use of oral combination of estradiol and a combination of vitamin D andcalcium is already known in the treatment of osteoporosis. Vitamin D usein this case is clearly intended to regulated calcium homeostasis andbone turnover.

Effect of a combination of oral raloxifene (60 mg/day) in associationwith 400 IU vitamin D and 600 mg of calcium on vaginal maturation indexand urogenital symptoms has been evaluated in postmenopausalosteoporotic women, by Zeyneloglu et al. These authors demonstrated animprovement in the vaginal maturation index and in the vaginal pH after3 months of a treatment with a raloxifen-based regimen, as compared witha 3 months treatment with a risedronate-based regimen, both regimencomprising a daily administration of 400 UI of vitamin D (Zeyneloglu H Bet al., Fertil Steril. 2007 August; 88(2):530-2).

Also, vaginal combination of high dose estriol (0.5 mg) and vitamin D(12500 IU) has been evaluated for the treatment of stress incontinence.The combination administered three times a week for six weeks increasedvitamin D serum level and brought a partial improvement on incontinencesymptoms to therapy with single estriol. The study was small and notconsidered representative by the authors. No effect on vaginalepithelium is mentioned. (Schulte-Uebbing C et al., Dermatoendocrinol.2016 Apr 19; 8(1):el 079359.

There is a need in the art for further pharmaceutical compositions thatare effective and endowed with a high level of safety for preventing ortreating vaginal atrophy.

SUMMARY OF THE INVENTION

This invention relates to a vaginal composition comprising a combinationof estrogen and vitamin D or a vitamin D analog at a daily dosagedelivery of (i) estrogen ranging from 1 μg to 100 μg estrogen ofestradiol equivalent and (ii) vitamin D or analog ranging from 7.5 μg to100 μg of vitamin D equivalent.

In some preferred embodiments, the daily dosage delivery of estrogenranges from 1 μg to 70 μg.

In some preferred embodiments, the daily dosage delivery of estrogenranges from 1 μg to 10 μg.

In some embodiments, the said vaginal composition comprises acombination of estradiol and calcitriol at a daily dosage delivery of(i) estradiol ranging from 1 μg to 10 μg and (ii) calcitriol rangingfrom 0.25 μg to 1 μg.

In some embodiments, the said vaginal composition has a liquid, solid orsemi-solid presentation.

In some embodiments, the said vaginal composition is a cream or a gelcomposition.

In some embodiments, the said vaginal composition is presented as dailyunit dosage form selected in a group comprising a capsule, an ovule, atablet and a suppository.

In some embodiments, the said vaginal composition is comprised in adelivery device selected in a group comprising a transmucosal device anda vaginal ring.

In some embodiments of the said vaginal composition, the said estrogenis estradiol or a derivative thereof.

In some embodiments of the said vaginal composition, the said vitamin Danalog is selected in a group comprising calcitriol and calcipotriol.

This invention also concerns a composition comprising a combination ofestrogen and vitamin D or vitamin D analog at a daily dosage delivery of(i) estrogen ranging from 1 μg to 100 μg estrogen of estradiolequivalent and (ii) vitamin D or analog ranging from 7.5 μg to 100 μg ofvitamin D equivalent for its use as a vaginal pharmaceuticalcomposition.

In some preferred embodiments of the use described above, the dailydosage delivery of estrogen ranges from 1 μg to 70 μg.

In some preferred embodiments of the use described above, the dailydosage delivery of estrogen ranges from 1 μg to 10 μg.

In some embodiments of the use described above, the said compositioncomprises a combination of estradiol and calcitriol at a daily dosagedelivery of (i) estradiol ranging from 1 μg to 10 μg and (ii) calcitriolranging from 0.25 μg to 1 μg.

This invention also pertains to a composition comprising a combinationof estrogen and vitamin D or vitamin D analog at a daily dosage deliveryof (i) estrogen ranging from 1 μg to 100 μg estrogen of estradiolequivalent and (ii) vitamin D or analog ranging from 7.5 μg to 100 μg ofvitamin D equivalent for its use for preventing or treating vaginalatrophy.

In some preferred embodiments of the use described above, the dailydosage delivery of estrogen ranges from 1 μg to 70 μg.

In some preferred embodiments of the use described above, the dailydosage delivery of estrogen ranges from 1 μg to 10 μg.

In some embodiments of the use described above, the said compositioncomprises a combination of estradiol and calcitriol at a daily dosagedelivery of (i) estradiol ranging from 1 μg to 10 μg and (ii) calcitriolranging from 0.25 μg to 1 μg.

This invention also relates to a method for preventing or treatingvaginal atrophy comprising a step of administering the vaginalcomposition described throughout the present specification to a woman inneed thereof.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a graph of body weight evolution. FIG. 1 depicts theevolution of body weight post-ovariectomy and during the 6-weektreatment period. Ordinate: Body weight, as expressed in grams.Abscissa: time period following ovariectomy, as expressed in days. Dataare represented as mean values +/− SEM, using the two-way ANOVA test.Although the curves corresponding to the various experimental conditionsmay be difficult to identify in the black and white FIG. 1 , the resultsof FIG. 1 show that there is no statistically significant differences inthe body weight between the different experimental conditions.

FIG. 2 illustrates a graph of the epithelium thickness. The epitheliumthickness values are reported as the percentage of increase as comparedwith the placebo conditions following a 6-week treatment period for eachof the experimental groups. Ordinate: epithelium thickness, as expressesin percentage increase versus placebo. Bars from the left to the rightof FIG. 2 : Estradiol 0.4 μg (n=7); Calcitriol 0.006 μg (n=6);Calcitriol 0.08 μg (n=7); Calcitriol 0.1 μg (n=6); Estradiol 0.2μg+Calcitriol 0.1 μg (n=7); Estradiol 0.2 μg+Calcitriol 0.08 μg (n=7);Estradiol 0.4 μg+Calcitriol 0.006 μg (n=7); Estradiol 0.02 μg+Calcitriol0.006 μg (n=7); Estradiol 0.02 μg+0.075 μg (n=8). Data are representedas the mean value+/− SEM. “****” means p<0.0001 versus placeboconditions using the Dunnett's multiple comparisons post-hoc testthrough the one-way ANOVA test. Dashed line: mean epithelium thicknessvalue of the animal group that has received placebo. In FIG. 2 ,significant results denoted “****” were calculated for the followingconditions: Estradiol 0.4 μg: Estradiol 0.2 μg+Calcitriol 0.1 μg;Estradiol 0.2 μg+Calcitriol 0.08 μg; Estradiol 0.4 μg+Calcitriol 0.006μg: Estradiol 0.02 μg+Calcitriol 0.006 μg; and Estradiol 0.02 μg+0.075μg.

FIG. 3 illustrates photographs of histological vaginal tissue slices (i)in placebo experimental conditions (FIG. 3A), (ii) animals treated with0.02 μg estradiol (FIG. 3B), (iii) animals treated with 0.08 μgestradiol (FIG. 3C) and (iv) animals treated with 0.4 μg estradiol (FIG.3D).

FIG. 4 illustrates the effect of the various treatment conditions on thematuration status of the vaginal tissue, as illustrated by thepercentage of cornified cells. Data (ordinate values) are presented asthe change from the baseline in the percentage of cornified cellsfollowing a 6-week treatment period. Data are presented as the meanvalues +/− SEM. “*” means p<0.05; “**” means p<0.01; “***” meansp<0.001. Statistical significance was determined by using Dunnett'smultiple comparisons post-hoc test following one-way ANOVA test. Barsform the left to the right of the FIG. 4 : Placebo (n=7); Estradiol 0.4μg (“***”; n=7); Calcitriol 0.006 μg (n=7); Calcitriol 0.08 μg (n=7);Calcitriol 0.1 μg (n=6); Estradiol 0.2 μg+Calcitriol 0.1 μg (p=0.051;n=8); Estradiol 0.2 μg+Calcitriol 0.08 μg (“*”; n=8); Estradiol 0.4μg+Calcitriol 0.006 μg (“*”; n=7); Estradiol 0.02 μg+Calcitriol 0.006 μg(“**”; n=8); Estradiol 0.02 μg+Calcitriol 0.075 μg (“***”; n=8).

FIG. 5 illustrates a graph depicting the effect of the various treatmentconditions on the maturation status of the vaginal tissue, asillustrated by the percentage of leukocytes. Data (ordinate values) arepresented as the change from the baseline in the percentage of cornifiedcells following a 6-week treatment period. Data are presented as themean values +/− SEM. “*” means p<0.05. Statistical significance wasdetermined by using Dunnett's multiple comparisons post-hoc testfollowing one-way ANOVA test. Bars form the left to the right of theFIG. 4 : Placebo (n=7); Estradiol 0.4 μg (n=7); Calcitriol 0.006 μg(n=7); Calcitriol 0.08 μg (n=7); Calcitriol 0.1 μg (n=6); Estradiol 0.2μg+Calcitriol 0.1 μg (n=8); Estradiol 0.2 μg+Calcitriol 0.08 μg (n=8);Estradiol 0.4 μg+Calcitriol 0.006 μg (n=7); Estradiol 0.02 μg+Calcitriol0.006 μg (n=8); Estradiol 0.02 μg+Calcitriol 0.075 μg (n=8).

FIG. 6 illustrates the calcium plasma level in the various treatmentconditions. Ordinate: calcium plasma level values, as expressed asmmol/L. From the left to the right of FIG. 6: Placebo (n=7); Calcitriol0.006 μg (n=7); Calcitriol 0.08 μg (“****”; n=7); Calcitriol 0.1 μg(“****”; n=7). Data are presented as the mean values +/− SEM. “****”means p<0.001 versus placebo. Statistical significance was determined byusing Dunnett's multiple comparisons post-hoc test following one-wayANOVA test.

DETAILED DESCRIPTION OF THE INVENTION

Any citation mentioned herein is incorporated by reference.

This invention relates to an estrogen-based composition for preventingor treating vaginal atrophy, and especially post-menopausal vaginalatrophy.

The present inventors have performed an extensive research with the aimof conceiving a pharmaceutical composition for preventing or treatingvaginal atrophy with the aim of obtaining a composition that shall be aseffective as the known compositions in its preventive or curativeeffect, and that shall be safer than the known compositions.

As described in detail in the present specification, the inventors haveconceived a specific pharmaceutical composition which is endowed with alocal action of the selected estrogen on the vaginal mucosa and which isbelieved to possess a limited or even no estrogen systemic action, andwhich is thus safer than the known pharmaceutical compositions.

The present inventors have conceived an estrogen-based pharmaceuticalcomposition comprising vitamin D or a vitamin D analog, whichpharmaceutical composition is under a physical form which is suitablefor a local release of the active ingredients to the vaginal mucosa.

According to a first aspect, a pharmaceutical composition according tothe invention releases locally an estrogen active ingredient at a dailyamount which is lower than the daily amount which is described in the anas an effective dose for preventing or treating vaginal atrophy,especially vaginal atrophy in post-menopausal women.

As it is shown in the examples herein, a pharmaceutical compositionaccording to the invention, when administered locally to individualsaffected with vaginal atrophy, allows reversing vaginal atrophy byincreasing the thickness of the vaginal epithelium and especially byincreasing the maturation status of the vaginal epithelium, asillustrated notably by the high increase of the cornified cells contentcontained therein.

According to a further aspect, a pharmaceutical composition according tothe invention releases locally vitamin D or vitamin D analog activeingredient at a daily amount which is at most the highest amount whichis recommended by the health agencies, which includes the Food and DrugAdministration in the US and the European Food Safety Authority (EFSAJournal 2012; 10(7):2813).

According to a still further aspect, a pharmaceutical compositionaccording to the invention is at least as effective for preventing ortreating vaginal atrophy as known estrogen-based compositions althoughthe said pharmaceutical composition comprises a very low amount of thesaid estrogen active ingredient.

According to a yet further aspect, a pharmaceutical compositionaccording to the invention, because it contains a very low amount of anestrogen active ingredient, is believed to be endowed with a decreasedrisk of side effects, and especially with a decreased risk ofhyperplasia or carcinoma, in comparison with the known compositions fortreating vaginal atrophy, including the known compositions for locallytreating vaginal atrophy. Indeed, because a vaginal compositionaccording to the invention comprises a very low amount of estrogen, thena risk of side effects caused by systemic passage of estrogen is farlower than with the low dose estrogen compositions known in the art.

According to still a further aspect, a pharmaceutical compositionaccording to the invention, because it contains at most the highestamount of vitamin D or vitamin D analog active ingredient recommended bythe health agencies, is believed to be endowed with a decreased risk ofside effects, and especially with a decreased risk of hypercalcemia.

The present invention relates to a vaginal composition comprising acombination of estrogen and vitamin D or a vitamin D analog at a dailydosage delivery of (i) estrogen ranging from 1 μg to 100 μg estrogen ofestradiol equivalent and (ii) vitamin D or analog ranging from 7.5 μg to100 μg of vitamin D equivalent.

In some preferred embodiments, the daily dosage delivery of estrogenranges from 1 μg to 70 μg.

In some other preferred embodiments, the daily dosage delivery ofestrogen ranges from 1 μg to 10 μg.

In some embodiments, the said vaginal composition comprises acombination of estradiol and calcitriol at a daily dosage delivery of(i) estradiol ranging from 1 μg to 10 μg and (ii) calcitriol rangingfrom 0.25 μg to 1 μg.

In some embodiments, the vaginal composition comprises a combination ofestrogen and vitamin D or a vitamin D analog at a daily dosage deliveryof (i) estrogen ranging from 2 μg to 10 μg estrogen of estradiolequivalent and (ii) vitamin D or analog ranging from 7.5 μg to 100 μg ofvitamin D equivalent.

As used herein, the expression “estrogen of estradiol equivalent” meansan amount of a selected estrogen which is expressed as the equivalentamount of estradiol. Estrogen equivalency is described elsewhere in thepresent specification.

As used herein, the expression “of vitamin D equivalent” means an amountof a selected vitamin D analog which is expressed as the equivalentamount of vitamin D. Vitamin D equivalency is described elsewhere in thepresent specification.

As used herein, the expression “daily dosage delivery” may be usedinterchangeably with the expression “daily delivery amount”. Theseexpressions mean the amount of the considered active ingredient (i.e.estrogen and vitamin D or vitamin D analog, respectively) which isreleased by the vaginal composition according to the invention. In mostembodiments, the amount of an active ingredient released by a vaginalcomposition according to the invention is also the amount of the saidactive ingredient which is comprised in the said composition.

In embodiments of a vaginal composition according to the invention whichare conceived for a daily administration of the said composition, the“daily dosage delivery” or “daily delivery amount” of a given activeingredient comprised therein (i.e. estrogen and vitamin D or vitamin Danalog, respectively) is the amount of the said active ingredientcomprised in the dosage unit which is to be administered.

In other embodiments of a vaginal composition according to the inventionwhich are conceived for releasing the active ingredients for a period oftime of two days or more, then the “daily dosage delivery” or “dailydelivery amount” of a given active ingredient comprised therein (i.e.estrogen and vitamin D or vitamin D analog, respectively) is the amountof the said active ingredient, comprised in the dosage unit, which isreleased daily by the said vaginal composition. The one skilled in theart readily understands that a vaginal composition aimed at releasing agiven daily amount of an active ingredient during a period of time of,e.g., seven days, shall comprise an amount of the said active ingredientwhich is at least seven times the said given daily amount.

The expression “of estradiol equivalent” means that the specified amountof estrogen is expressed as the amount in estradiol equivalent value,the said equivalency being described elsewhere in the presentspecification.

The expression “of vitamin D equivalent” means that the specified amountof vitamin D or analog is expressed as the amount in vitamin Dequivalent value, the said equivalency being described elsewhere in thepresent specification.

A daily dosage of vitamin D ranging from 7.5 μg to 100 μg consists of adaily dosage ranging from 300 IU to 4000 IU of vitamin D, as it is wellknown in the art.

In some embodiments of the vaginal composition wherein vitamin D oranalog is calcitriol or calcipotriol, then the said vaginal compositioncomprises calcitriol or calcipotriol at a daily dosage delivery rangingfrom 0.075 μg to 1.000 μg, which corresponds to 7.5 μg to 100 μg ofvitamin D equivalent.

A daily dosage of calcitriol or calcipotriol ranging from 0.075 μg to1.00 μg encompasses a daily dosage of calcitriol or calcipotriol rangingfrom 0.25 μg to 1.00 μg, and a daily dosage of calcitriol orcalcipotriol ranging from 0.5 μg to 1.00 μg.

In preferred embodiments, the combination of estrogen and vitamin D orvitamin D analog is the sole combination of active ingredients inducingan effect on vaginal atrophy that is comprised in a vaginal compositionaccording to the invention.

Illustratively, a vaginal composition does not comprise any selectiveestrogen receptor modulator compound (also termed “SERM”), such asraloxifene, tamoxifen, clomifene, ormeloxifene, toremifene, lasofoxifeneor ospermifene.

In most preferred embodiments, estrogen and vitamin D or vitamin Danalog are the sole active ingredients comprised in a vaginalcomposition according to the invention.

A vaginal composition according to the invention may comprise one ormore estrogens.

A vaginal composition comprising estrogen at a daily dosage ranging from2 μg to 10 μg of estradiol equivalent comprises estrogen at a dailydosage amount ranging from 0.029 μg/kg to 0.142 μg/kg of estradiolequivalent, based on a woman weighing 70 kg.

In some embodiments, the vaginal composition comprises estrogen at adaily dosage ranging from 1 μg to 10 μg of estradiol equivalentcomprises estrogen at a daily dosage amount ranging from 0.014 μg/kg to0.142 μg/kg of estradiol equivalent, based on a woman weighing 70 kg.

In some embodiments, the vaginal composition comprises estrogen at adaily dosage ranging from 1 μg to 100 μg of estradiol equivalentcomprises estrogen at a daily dosage amount ranging front 0.014 μg/kg to1.42 μg/kg of estradiol equivalent, based on a woman weighing 70 kg.

In most preferred embodiments, a vaginal composition according to theinvention comprises only one estrogen.

In some embodiments, a vaginal composition according to the inventioncomprises an amount of estrogen for a daily dosage delivery of an amountof estradiol equivalent ranging from 2 μg to 7.5 μg. According to theseembodiments, a vaginal composition according to the invention comprisesan amount of estradiol equivalent ranging from 0.029 μg/kg to 0.11μg/kg, based on a woman weighing 70 kg.

In some embodiments, a vaginal composition according to the inventioncomprises an amount of estrogen for a daily dosage delivery of an amountof estradiol equivalent ranging from 1 μg to 7.5 μg. According to theseembodiments, a vaginal composition according to the invention comprisesan amount of estradiol equivalent ranging from 0.014 μg/kg to 0.11μg/kg, based on a woman weighing 70 kg.

In some embodiments, a vaginal composition according to the inventioncomprises vitamin D or an analog thereof at a daily dosage delivery ofvitamin D or analog ranging from 7.5 μg to 100 μg of vitamin Dequivalent.

In some preferred embodiments, vitamin D analog is calcitriol. In theseembodiments, a vaginal composition according to the invention comprisescalcitriol at a daily dosage delivery* ranging from 0.075 μg to 1.000 μgof calcitriol. In some aspects of these preferred embodiments, a vaginalcomposition according to the invention comprises calcitriol at a dailydosage delivery ranging from 0.25 μg to 1.00 μg of calcitriol. In someother aspects of these preferred embodiments, a vaginal compositionaccording to the invention comprises calcitriol at a daily dosageranging from 0.50 μg to 1.00 μg of calcitriol.

Thus, in some preferred embodiments, a vaginal composition as describedherein comprises a combination of estradiol and calcitriol at a dailydosage delivery of (i) estradiol ranging from 1 μg to 10 μg and (ii)calcitriol ranging from 0.25 μg to 1 μg.

As used herein, a vaginal composition encompasses a pharmaceuticalcomposition for local administration of the active ingredients in viewof the release of these active ingredients at the expected amount at thevaginal mucosa.

In some embodiments of the vaginal composition according to theinvention, the said composition is under a pharmaceutical formcomprising the daily dosage amount of the combined estrogen and vitaminD or analog active ingredients.

In some other embodiments of the vaginal composition according to theinvention, the said composition is under a pharmaceutical formcomprising an amount of the combined estrogen and vitamin D or analogthat represents a plurality of daily dosage amounts, the saidpharmaceutical form releasing each day the required daily amount of thesaid combined active ingredients.

In all embodiments, a vaginal composition according to the inventioncomprises an amount of estrogen allowing the release of a daily amountof estrogen ranging from 1 μg to 100 μg estrogen of estradiolequivalent, which encompasses of a daily amount of estrogen ranging from1 μg to 7.5 μg estrogen of estradiol equivalent.

In some embodiments, the daily dosage delivery of estrogen ranges from 1μg to 70 μg.

In some other embodiments, the daily dosage delivery of estrogen rangesfrom 1 μg to 10 μg.

In some embodiments, a vaginal composition according to the inventioncomprises an amount of estrogen allowing the release of a daily amountof estrogen ranging from 2 μg to 10 μg estrogen of estradiol equivalent,which encompasses of a daily amount of estrogen ranging from 2 μg to 7.5μg estrogen of estradiol equivalent.

In all embodiments, a vaginal composition according to the inventioncomprises an amount of vitamin D or analog allowing the release of adaily amount of the said vitamin D or analog ranging from 7.5 μg to 100μg of vitamin D equivalent.

In some embodiments, a vaginal composition according to the inventioncomprises an amount of vitamin D or analog allowing the release of adaily amount of the said vitamin D or analog ranging from 7.5 μg to 100μg of vitamin D equivalent. In some embodiments, a vaginal compositionaccording to the invention comprises an amount of vitamin D or analogallowing the release of a daily amount of the said vitamin D or analogranging from 7.5 μg to 25 μg of vitamin D equivalent In someembodiments, a vaginal composition according to the invention comprisesan amount of vitamin D or analog allowing the release of a daily amountof the said vitamin D or analog ranging from 15 μg to 100 μg of vitaminD equivalent. In some embodiments, a vaginal composition according tothe invention comprises an amount of vitamin D or analog allowing therelease of a daily amount of the said vitamin D or analog ranging from15 μg to 25 μg of vitamin D equivalent

In some embodiments, a vaginal composition according to the inventioncomprises (i) an amount of estradiol allowing the release of a dailyamount of estradiol ranging from 1 μg to 10 μg. and (ii) an amount ofcalcitriol allowing the release of a daily amount of calcitriol rangingfrom 0.25 μg and 1.000 μg.

In all embodiments of a vaginal composition according to the invention,the combination of estrogen and vitamin D or vitamin D analog is furthercombined with one or more physiologically acceptable excipients.

In all embodiments of a vaginal composition according to the invention,the one or more physiological excipients are present in a quantitysufficient so as to amount at 100% of the weight of the vaginalcomposition, or alternatively at 100% of the volume of the vaginalcomposition, depending on the amount unit which is used.

In embodiments of a vaginal composition according to the inventionwherein the said composition is under a pharmaceutical form suitable fora daily administration, such as a pharmaceutical form selected in agroup comprising a cream, a gel, a suppository (such as an ovule, acapsule or a tablet), then a daily unit dosage of the said vaginalcomposition comprises (i) an estrogen at an amount ranging from 1 μg to100 μg of estradiol equivalent and (ii) vitamin D or analog ranging from7.5 μg to 100 μg of vitamin D equivalent. In some aspects of theseembodiments, a daily unit dosage of the said vaginal compositioncomprises (i) an estrogen at an amount ranging from 1 μg to 100 μg ofestradiol equivalent and (ii) vitamin D or analog ranging from 15 μg to100 μg of vitamin D equivalent. In some aspects of these embodiments, adaily unit dosage of the said vaginal composition comprises (i) anestrogen at an amount ranging from 1 μg to 100 μg of estradiolequivalent and (ii) vitamin D or analog ranging from 15 μg to 25 μg ofvitamin D equivalent. In some aspects of these embodiments, a daily unitdosage of the said vaginal composition comprises (i) an estrogen at anamount ranging from 1 μg to 10 μg of estradiol equivalent and (ii)vitamin D or analog ranging from 7.5 μg to 100 μg of vitamin Dequivalent. In some aspects of these embodiments, a daily unit dosage ofthe said vaginal composition comprises (i) an estrogen at an amountranging from 1 μg to 7.5 μg of estradiol equivalent and (ii) vitamin Dor analog ranging from 7.5 μg to 100 μg of vitamin D equivalent. In somefurther aspects of these embodiments, a daily unit dosage of the saidvaginal composition comprises (i) an estrogen at an amount ranging from1 μg to 10 μg of estradiol equivalent and (ii) vitamin D or analogranging from 15 μg to 100 μg of vitamin D equivalent. In still somefurther aspects of these embodiments, a daily unit dosage of the saidvaginal composition comprises (i) an estrogen at an amount ranging from1 μg to 7.5 μg of estradiol equivalent and (ii) vitamin D or analogranging from 15 μg to 100 μg of vitamin D equivalent. In yet somefurther aspects of these embodiments, a daily unit dosage of the saidvaginal composition comprises (i) an estrogen at an amount ranging from1 μg to 10 μg of estradiol equivalent and (ii) vitamin D or analogranging from 15 μg to 25 μg of vitamin D equivalent. In still somefurther aspects of these embodiments, a daily unit dosage of the saidvaginal composition comprises (i) an estrogen at an amount ranging from1 μg to 7.5 μg of estradiol equivalent and (ii) vitamin D or analogranging from 15 μg to 25 μg of vitamin D equivalent.

In some of these embodiments, a vaginal composition according to theinvention a daily unit dosage of the said vaginal composition comprises(i) estradiol at an amount ranging from 1 μg to 10 μg and (ii) calitriolor calcipotriol at an amount ranging from 0.075 μg to 1.000 μg. In someaspects of these embodiments, a daily unit dosage of the said vaginalcomposition comprises (i) an estradiol at an amount ranging from 1 μg to7.5 μg and (ii) calitriol or calcipotriol ranging from 0.075 μg to 1.00μg. In some further aspects of these embodiments, a daily unit dosage ofthe said vaginal composition comprises (i) an estradiol at an amountranging from 1 μg to 10 μg and (ii) calcitriol or calcipotriol rangingfrom 0.5 μg to 1.00 μg. In still some further aspects of theseembodiments, a daily unit dosage of the said vaginal compositioncomprises (i) an estradiol at an amount ranging from 1 μg to 7.5 μg and(ii) calitriol or calcipotriol ranging from 0.5 μg to 4.00 μg. In yetsome further aspects of these embodiments, a daily unit dosage of thesaid vaginal composition comprises (i) an estradiol at an amount rangingfrom 1 μg to 10 μg of and (ii) calitriol or calcipotriol ranging from0.25 μg to 1.00 μg. In still some further aspects of these embodiments,a daily unit dosage of the said vaginal composition comprises (i) anestradiol at an amount ranging from 1 μg to 7.5 μg and (ii) calitriol orcalcipotriol ranging from 0.25 μg to 1.00 μg. In yet some furtheraspects of these embodiments, a daily unit dosage of the said vaginalcomposition comprises (i) an estradiol at an amount ranging from 1 μg to10 μg of and (ii) calitriol or calcipotriol ranging from 0.5 μg to 1.00μg. In still some further aspects of these embodiments, a daily unitdosage of the said vaginal composition comprises (i) an estradiol at anamount ranging from 1 μg to 7.5 μg and (ii) calitriol or calcipotriolranging from 0.5 μg to 1.00 μg.

In some of these embodiments, a vaginal composition according to theinvention a daily unit dosage of the said vaginal composition comprises(i) estradiol at an amount ranging from 1 μg to 10 μg and (ii) calitriolat an amount ranging from 0.25 μg to 1.000 μg.

In embodiments of the vaginal composition according to the inventionwherein the said composition is under a pharmaceutical form suitable fora continuous release of the combined active ingredients during aplurality of days, such as about 15 days or about 30 days, such ascomprised in a transmucosal delivery system or in a vaginal ring, thenthe said vaginal composition comprises an amount of the combined activeingredients which is at minimum a multiple of the required daily dosagethat is released by the said delivery system or by the said vaginalring.

For example, in embodiments wherein the vaginal composition is comprisedin a delivery system, such as a transmucosal delivery system or avaginal ring, that shall remain in the vaginal cavity for a period oftime of 15 days, then the said vaginal composition may comprise atminimum a combination of estrogen and vitamin D or a vitamin D analog ata daily dosage delivery of (i) estrogen ranging from 15 μg to 1500 μgestrogen of estradiol equivalent and (ii) vitamin D or analog rangingfrom 112.5 μg to 1500 μg of vitamin D equivalent.

For example, in some other embodiments wherein the vaginal compositionis comprised in a delivery system, such as a transmucosal deliverysystem or a vaginal ring, that shall remain in the vaginal cavity for aperiod of time of 15 days, then the said vaginal composition maycomprise at minimum a combination of estrogen and vitamin D or a vitaminD analog at a daily dosage delivery of (i) estrogen ranging from 30 μgto 150 μg estrogen of estradiol equivalent and (ii) vitamin D or analogranging from 112.5 μg to 1500 μg of vitamin D equivalent.

In such embodiments, the amount of each of the combined activeingredients is preferably higher than the sole amount required for thetime period wherein the delivery system is located in the vaginalcavity, so as to ensure that the required daily amount of the combinedactive ingredients will be actually released and will remain as constantas possible during the whole said time period.

Estrogen

As used herein, “estrogen” encompasses estrogenic steroids selected in agroup comprising estradiol (17-β-estradiol), estradiol valerate,estradiol benzoate, estradiol 17β-cypionate, estradiol dipropionate,estradiol enanthate, estropipate, equilenin, equilin, estriol, estriolsuccinate, estrone, ethinyl estradiol, estetrol, quinestrol,quinestranol, conjugated estrogens (equine or synthetic), esterifiedestrogens, and mixtures thereof.

The estrogen may be selected, for example, from a group comprising ofethinyl estradiol, 17-[beta]-estradiol, conjugated estrogens, mestranol,estetrol, estrone and esters, prodrugs and salts thereof. An exemplaryester is estradiol acetate. Preferred salts of estrone include, but arenot limited to the sodium, sulfate and piperate salt.

For the conjugated estrogens, 1.25 mg conjugated estrogens is equivalentto a daily dose of 15 μg ethinyl estradiol. The most preferred estrogenis estradiol.

Determination of estradiol equivalent potency is well understood andreadily accomplished by those of ordinary skill in the art.Illustratively, for determining the estradiol equivalent value of anestrogen amount, the one skilled in the art may refer to the Europeanpatent application no EP 0253607 or to the PCT application no. WO2007/089733. Still illustratively, 30 μg of ethinyl estradiol is roughlyequivalent to 60 μg of mestranol or 2 mg of 17 beta estradiol.

In preferred embodiments of a vaginal composition according to theinvention, the estrogen is estradiol

The term “estradiol” refers to(17beta)-estra-1,3,5(10)-triene-3,17-diol. Estradiol is alsointerchangeably called 17beta-estradiol, oestradiol, or E2.

In some embodiments, the estradiol starting product for manufacturing avaginal composition according to the invention may be provided in ananhydrous or hemi-hydrate form. Indeed, the anhydrous form or thehemihydrate form can be substituted for the other by accounting for thewater or lack of water according to well-known and understood techniques

The term “solubilized estradiol” means that the estradiol or a portionthereof is solubilized or dissolved in one or more solubilizing agent(s)for preparing a vaginal composition disclosed herein. Solubilizedestradiol may include estradiol that is about 80% solubilized, about 85%solubilized, about 90% solubilized, about 95% solubilized, about 96%solubilized, about 97% solubilized, about 98% solubilized, about 99%solubilized or about 100% solubilized. In some embodiments, theestradiol is “fully solubilized” with all or substantially all of theestradiol being solubilized or dissolved in the solubilizing agent.Fully solubilized estradiol may include estradiol that is about 97%solubilized, about 98% solubilized, about 99% solubilized or about 100%solubilized. Solubility can be expressed as a mass fraction (% w/w,which is also referred to as wt %).

In some embodiments, estradiol is used in a micronized form. The term“micronized estradiol,” as used herein, include micronized micronizedestradiol having an X₅₀ particle size value below about 15 microns orhaving an X₉₀ particle size value below about 25 microns. The term “X₅₀”means that one-half of the particles in a sample are smaller in diameterthan a given number. For example, micronized estradiol having an X₅₀ of5 microns means that, for a given sample of micronized estradiol,one-half of the particles have a diameter of less than 5 microns.Similarly, the term “X₉₀” means that ninety percent (90%) of theparticles in a sample are smaller in diameter than a given number.

Vitamin D and Vitamin D Analogs

As intended herein a “vitamin D analog” is a compound that binds to thevitamin D receptor (VDR).

Tests for determining the ability of a vitamin D analog or of a vitaminD receptor modulator to bind to the vitamin D receptor are well known tothe person skilled in the art. Preferably, this ability may be evaluatedby measuring the specific binding of the said analog or of the vitamin Dreceptor modulator on a cell extract. For example, in a typical bindingexperiment, soluble cell extract obtained by sonication is incubatedwith increasing concentration of vitamin D analog or of vitamin Dreceptor modulator. Bounds and free analogs can be separated by thehydroxylapatite method. Specific binding may be calculated bysubtracting non-specific binding obtained in the presence of an excess1,25-(OH)2D3 from the total binding measured in absence of 1,25-(OH)2D3(Skowronski et al. (1995) Endocrynology 136(1):20-26).

As used herein, active form of vitamin D is known as1α,25-dihydroxyvitamin D3 (1,25(OH)₂D3 or calcitriol).

As used herein, vitamin D analogs encompass cholecalciferol (also termedas vitamin D3) and ergocalciferol (also termed as vitamin D2) and theirmetabolites as well as synthetic cholecalciferol and ergocalciferolanalogs. These synthetic cholecalciferol and ergocalciferol analogscomprise such categories of compounds as the 5,6 trans-cholecalciferolsand 5,6 trans-ergocalciferols, the fluorinated cholecalciferols, theside chain homologated cholecalciferols and side chain homologated 22cholecalciferols the side chain truncated cholecalciferols, the 19-norcholecalciferols and ergocalciferols and the 10,19-dihydrovitamin Dcompounds. Some specific examples include but are not limited tocholecalciferol (vitamin D3, calciol), ergocalciferol (vitamin D2,ercalciol), alphacalcidol (1 alpha-hydroxy vitamin D3), calcidiol(25-hydroxyvitamin D3), calcitriol (1,25-dihyroxyvitamin D3),calcifediol, calcipotriol (calcipotriene), 22-oxacalcitriol (OCT),paricalcitol (19-Nor-1 alpha,25-dihydroxyvitamin D2), doxercalciferol,eldecalcitol, dihydrotachysterol-2 (DHT-2).

The vitamin D analog 22-oxacalcitriol (OCT) differs from 1,25(OH)₂D3 byvirtue of an oxygen atom replacing carbon-22 on the side chain.

Paricalcitol is a vitamin D2 derived sterol lacking the carbon-19methylene group found in all natural vitamin D metabolites.

Doxercalciferol (1α-hydroxyvitamin D2), like alfacalcidol(1α-hydroxyvitamin D3), is a pro-drug which is hydroxylated in the liverto 1α,25(OH)2D2.

Dihydrotachysterol-2 (DHT-2), hydroxylated in vivo to 25(OH)DHT2 is alsoof interest for a vaginal composition according to the invention.

Vitamin D and vitamin D analogs are well known by the one skilled in theart, as well as their respective methods of synthesis. Further, vitaminD and analogs thereof are commercially available to the one skilled inthe art.

Pharmaceutical Forms

Vaginal compositions according to the invention may be provided invarious pharmaceutical forms that are suitable tor the local delivery ofthe combined estrogen and vitamin D or vitamin D analog to the vaginalmucosa.

Different pharmaceutical forms for administering drugs intravaginallyhave been described, such as suppositories (such as ovules, capsules andtablets), solutions, creams, foams, gels, films and vaginal rings.Intravaginal delivery systems are described in patents and patentapplications such as U.S. Pat. No. 6,086,908, no US 2005/0276836, U.S.Pat. No. 6,086,909, no EP 0889724, U.S. Pat. No. 7,004,171, no US2008/193428 and U.S. Pat. No. 5,989,581, wherein intravaginal releasesystems are described and between the alternatives vaginal rings arementioned.

A vaginal composition according to the invention may be also provided insyringe-like applicators for local vaginal administration.

Vaginal cream compositions are for example described in the patentapplications and patents no. EP 2849735, WO 2006/023496 and U.S. Pat.No. 5,514,698. Vaginal tablet compositions are for example described inthe patent applications and patents no. WO 2015/135915, US 2011/159091,U.S. Pat. No. 3,062,715 and EP 0900564.

Vaginal film compositions are for example described in the patentapplications no WO 2004/103232 and US 2005/070501.

A vaginal composition according to the invention may be provided as avaginal cream or gel, a vaginal suppository (such as a vaginal ovule, avaginal capsule, a vaginal tablet), or may be comprised in a vaginaldelivery system such as a transmucosal device, a sponge-like device or avaginal ring.

As used herein, a “vaginal suppository” encompasses a vaginal ovule, avaginal tablet or a vaginal capsule.

Vaginal suppositories are known in the art, and may be made of glycerin,fatty acids, and similar type substances that dissolve at bodytemperature. As the suppository dissolves, the combined estrogen andvitamin D or vitamin D analog will be released.

In some embodiments, a vaginal suppository according to the inventioncomprises an inert vehicle. As used herein the term “inert vehicle”refers to a vehicle which brings the active substance, in this caselactic acid or a salt thereof in contact with the vaginal tissue. Theinert vehicle enables the fabrication of a suppository which isrelatively solid at room temperature and in a dry environment, but whichmelts at body temperature and in contact with body fluids. Any inertvehicle which has the above characteristics may be used, but typicallypolyethylene glycol (PEG) is used. In embodiments of the invention, theinert vehicle comprises polyethylene glycol 600 and polyethylene glycol4000. The ratio between PEG 600 and PEG 4000 may be varied which allowsfor the dissolution time of the vaginal suppositories to be varied. Theproperties of the inert vehicle determines the dissolution time of thevaginal suppositories in the vagina.

In some embodiments, a vaginal composition according to the invention isunder the form of an ovule, e.g. a pharmaceutical form wherein the saidcombined estrogen and vitamin D or vitamin D analog are comprised withina lipophilic suppository base. Illustratively, the one skilled in theart may prepare a vaginal composition according to the inventionstarting from a lipophilic suppository base such as the startingsuppository composition marketed under the name Ovucire® by the Frenchcompany Gatefossé.

An ovule of the present invention may be prepared according to a methodcomprising the steps of:

-   -   a) melting the Ovucire® mixture at a temperature of 45-80° C.;    -   b) precooling the mixture by slowly stirring until a mass        temperature of 40-60° C. is reached and then adding a volume of        50 μl of placebo or the active ingredient(s) liquid material        under vigorous stirring;    -   d) keeping stirring slowly until the mold temperature reaches        35-50° C.;    -   e) filling the molds with the mass;    -   f) cooling the ovules in the molds; and    -   g) scaling the molds.

A vaginal composition according to the invention may be provided underthe form of a vaginal cream or a vaginal gel. A “gel” is a colloid inwhich a disperse phase combines with a dispersion medium to produce ajelly-like, solid or semi-solid material. Although a variety ofcompounds may be employed, water is usually employed as the dispersionmedium for the gel to optimize biocompatibility. Other possibledispersion mediums include non-aqueous solvents, including glycols, suchas propylene glycol, butylene glycol, triethylene glycol, hexyleneglycol, polyethylene glycols, ethoxydiglycol, and dipropyleneglycol;alcohols, such as ethanol, n-propanol, and isopropanol; triglycerides;ethyl acetate; acetone; triacetin; and combinations thereof. Typically,the dispersion medium (e.g., water) constitutes greater than about 75wt/vol %, in some embodiments greater than about 90 wt/vol %, and insome embodiments, from about 95 wt/vol % to about 99 wt/vol % of thevaginal treatment composition. As used herein, the designation “wt/vol%” or “wt/vol” or refers to the value obtained by dividing the weight ofa substance (in grams) by the volume of the solution (in milliliters),and then multiplying by 100.

A vaginal composition according to the invention may be comprised inspecific delivery systems such as transmucosal delivery systems or alsoin vaginal ring delivery systems.

As used herein, the term “transmucosal” refers to delivery,administration or application of a drug by means of direct contact withskin or mucosa. Such delivery, administration or application is alsoknown us transmucosal. As used herein, “mucosal” includes mucosa, whichincludes vaginal mucosa.

As used herein, “transmucosal drug delivery system” refers to a system(e.g., a device) comprising a composition that releases the combinationof estrogen and vitamin D or vitamin D analog upon application to thevaginal mucosa. A transmucosal drug delivery system may comprise abacking layer, a drug-containing layer, and a release liner layer. Insome embodiments, the transdermal drug delivery system is asubstantially non-aqueous, solid form, capable of conforming to thesurface with which it comes into contact, and capable of maintainingsuch contact so as to facilitate topical application on the vaginalmucosa without adverse physiological response, and without beingappreciably decomposed by aqueous contact during topical application toa subject. Many such systems are known in the art and commerciallyavailable, such as transmucosal drug delivery patches. As describedbelow, in one embodiment, the transmucosal drug delivery systemcomprises a drug-containing polymer matrix that comprises apressure-sensitive adhesive or bioadhesive, and is adopted tor directapplication to a user's (e.g., a subject's) skin. In other embodiments,the polymer matrix is non-adhesive and may be provided with separateadhesion means (such as a separate adhesive layer) for application andadherence to the user's vaginal mucosa. Transmucosal delivery systemsare notably described in the patent applications and patents such as CN102641548, WO 2005/016321, WO 2004/067063, U.S. Pat. No. 5,204,108. US2004/043071 and JP 3 705 620.

As used herein, “polymer matrix” refers to a polymer composition whichcontains one or more drugs. In some embodiments, the matrix comprises apressure-sensitive adhesive polymer or a bioadhesive polymer. In otherembodiments, the matrix does not comprise a pressure-sensitive adhesiveor bioadhesive. As used herein, a polymer is an “adhesive” if it has theproperties of an adhesive per se, or if it functions as an adhesive bythe addition of tackifiers, plasticizers, crosslinking agents or otheradditives. Thus, in some embodiments, the polymer matrix comprises apressure-sensitive adhesive polymer or a bioadhesive polymer, withestrogen dissolved or dispersed therein. The polymer matrix also maycomprise tackifiers, plasticizers, crosslinking agents or otheradditives.

A vaginal composition according to the invention may also be comprisedin a vaginal ring.

Many of vaginal ring devices known by the one skilled in the an may beused for the purpose of the present invention.

In some embodiments, a vaginal ring delivery system for use with avaginal composition according to the invention may be manufactured as itfollows. A homogeneous blend of all the ingredients to be injected intothe ring molds was prepared. First the required amounts of eachingredient were weighed: Polymer A, release modifier agent, ifapplicable, and meloxicam. These ingredients were mixed untilhomogenization and the polymer B was added under constant mixing. Themixture was injected into ring molds at room temperature and then keptin an oven at 105° C. for 1 hour. Subsequently molds were cooled and theformed rings were disassembled from their respective molds obtaining thefinal product.

Vaginal rings and methods for preparing the same are notably describedin the patent applications and patents WO 2016/054002, EP 2 799042, US2015/328319. WO 2011/011099, WO2013/098591 and US 2013/269706.

Methods of Prevention and Treatment of Vaginal Atrophy

The present invention further relates to a method for preventing ortreating vaginal atrophy, which method comprises a step of administeringa vaginal composition as described in the present specification to awoman in need thereof.

Thus, this invention to a method for preventing or treating vaginalatrophy comprising a step of administering, to a woman in need thereof,a vaginal composition comprising a combination of estrogen and vitamin Dor a vitamin D analog at a daily dosage delivery of (i) estrogen rangingfrom 1 μg to 100 μg estrogen of estradiol equivalent and (ii) vitamin Dor analog ranging from 7.5 μg to 100 μg of vitamin D equivalent.

In some embodiments, the daily dosage delivery of estrogen ranges from 1μg to 70 μg.

In some other embodiments, the daily dosage delivery of estrogen rangesfrom 1 μg to 10 μg.

In some other embodiments of the method, the said vaginal compositioncomprises estradiol and calcitriol at a daily dosage delivery of (i)estradiol ranging from 1 μg to 10 μg and (ii) calitriol ranging from0.25 μg to 1.000 μg.

In some other embodiments, the invention also relates to a method forpreventing or treating vaginal atrophy comprising a step ofadministering, to a woman in need thereof, a vaginal compositioncomprising a combination of estrogen and vitamin D or a vitamin D analogat a daily dosage delivery of (i) estrogen ranging from 2 μg to 10 μgestrogen of estradiol equivalent and (ii) vitamin D or analog rangingfrom 7.5 μg to 100 μg of vitamin D equivalent.

In some embodiments of the said composition, the said estrogen isestradiol.

In some embodiments of the said composition, the said vitamin D orvitamin D analog is selected in a group comprising calcitriol andcalcipotriol.

In some embodiments of the method, the said vaginal composition isliquid, solid or semi-solid.

In some embodiments, the said vaginal composition is a cream or a gelcomposition.

In some embodiments, the said vaginal composition is presented as dailyunit dosage form selected in a group comprising a capsule, an ovule, atablet and a suppository.

In some embodiments, the said vaginal composition is comprised in adelivery device selected in a group comprising a transmucosal device anda vaginal ring.

In the embodiments of the method wherein the vaginal composition isunder the form of a cream, a gel or a suppository (such as a capsule, anovule or a tablet), the said composition is preferably administered atleast once daily, which includes one daily administration of the vaginalcomposition.

In the embodiments of the method wherein the vaginal composition iscomprised in a transmucosal delivery system or a vaginal ring, the saidtransmucosal delivery system or the said vaginal ring is placed in thevaginal cavity at defined periodicity and the vaginal composition iscontinuously released from the transmucosal delivery system or vaginalring at a rate which is appropriate for releasing the combined estrogenand vitamin D or vitamin D analog at a daily dosage delivery of (i)estrogen ranging from 1 μg to 10 μg estrogen of estradiol equivalent and(ii) vitamin D or analog ranging from 7.5 μg to 100 μg of vitamin Dequivalent.

In some of these embodiments, the vaginal composition is comprised in atransmucosal delivery system or a vaginal ring, the said transmucosaldelivery system or the said vaginal ring is placed in the vaginal cavityat defined periodicity and the vaginal composition is continuouslyreleased from the transmucosal delivery system or vaginal ring at a ratewhich is appropriate for releasing the combined estradiol and calcitriolat a daily dosage delivery of (i) estradiol ranging from 1 μg to 10 μgand (ii) calcitriol ranging from 0.25 μg to 1 μg.

In the embodiments of the method wherein the vaginal composition iscomprised in a transmucosal delivery system or a vaginal ring, the saidtransmucosal delivery system or the said vaginal ring is placed in thevaginal cavity at defined periodicity and the vaginal composition iscontinuously released from the transmucosal delivery system or vaginalring at a rate which is appropriate for releasing the combined estrogenand vitamin D or vitamin D analog at a daily dosage delivery of (i)estrogen ranging from 2 μg to 10 μg estrogen of estradiol equivalent and(ii) vitamin D or analog ranging from 7.5 μg to 100 μg of vitamin Dequivalent.

In some of the embodiments of the method wherein the vaginal compositionis comprised in a transmucosal delivery system or a vaginal ring, thesaid transmucosal delivery system or the said vaginal ring is placed inthe vaginal cavity at defined periodicity and the vaginal composition iscontinuously released from the transmucosal delivery system or vaginalring at a rate which is appropriate for releasing the combined estradioland calcitriol at a daily dosage delivery of (i) estradiol ranging from1 μg to 10 μg and (ii) calcitriol ranging from 0.25 μg to 1 μg ofvitamin D equivalent.

Uses of the Vaginal Composition Disclosed herein

The invention further relates to the use of a composition comprising acombination of estrogen and vitamin D or vitamin D analog at a dailydosage delivery of (i) estrogen ranging from 1 μg to 100 μg estrogen ofestradiol equivalent and (ii) vitamin D or analog ranging from 7.5 μg to100 μg of vitamin D equivalent for treating vaginal atrophy.

In some embodiments, the daily dosage delivery of estrogen ranges from 1μg to 70 μg.

In some embodiments, the daily dosage delivery of estrogen ranges from 1μg to 10 μg.

The invention further relates to the use of a composition comprising acombination of estradiol and calcitriol at a daily dosage delivery of(i) estradiol ranging from 1 μg to 100 μg and (ii) calcitriol rangingfrom 0.075 μg to 1 μg as a vaginal pharmaceutical composition.

In some embodiments, the daily dosage delivery of estrogen ranges from 1μg to 70 μg.

In some embodiments, the daily dosage delivery of estrogen ranges from 1μg to 10 μg.

The invention further relates to the use of a composition comprising acombination of estradiol and calcitriol at a daily dosage delivery of(i) estradiol ranging from 1 μg to 100 μg and (ii) calcitriol rangingfrom 0.075 μg to 1 μg for treating vaginal atrophy.

In some embodiments, the daily dosage delivery of estrogen ranges from 1μg to 10 μg.

EXAMPLES Example 1: Evaluation of the Effect of a Combination ofEstradiol and Vitamin D on Vaginal Atrophy in an Experimental Model ofMenopause

The effect of the combination of a low dose estrogen with a dose ofvitamin D on vaginal atrophy is explored by using a recognizedexperimental model of menopause, namely in the experimental model ofovariectomized rodent, i.e. in ovariectomized rat and ovariectomizedmouse.

A. Animal Model of Menopause and VVA: Ovariectomized Rodent (Rat orMice)

Ovariectomized rat is an animal model of estrogen deficiency that hasbeen used to evaluate vaginal effects of different products such asospemifene or DHEA. Dose response of ospemifene in the rat wasconsistent with that observed in clinical studies, showing that thisanimal model is a highly predictive model of ospemifene activity inpostmenopausal vulvovaginal atrophy (Unkila M et al., J Steroid BiochemMol Biol. 2013 November; 138:107-15.). Vaginal effects of intravaginalapplication of DHEA has also been studied in this animal model (Berger Let al., J Steroid Biochem Mol Biol. 2008 March; 109(1-2):67-80. Berger Let al., J Steroid Biochem Mol Biol. 2005 Jul; 96(2):201-15.).

B. Markers of Vaginal Atrophy

Occurrence of vaginal atrophy as well as degree of vaginal atrophy shallbe assessed through the use of a plurality of markers which are thefollowing:

-   -   vaginal epithelium thickness and morphology after chronic        treatment;    -   measurement of vaginal tissue wet weight as an index of tissue        atrophy    -   measurement of vaginal pH, preferably by using a pH indicator        strip before and after chronic treatment,    -   measurement of the vaginal maturation index (VMI), which        measurement is performed on vaginal smears before and after        chronic treatment.

C. Marker of Systemic Effect

Systemic effect of a local hormonal treatment shall be assessed bymeasurement of the uterus weight after chronic treatment.

C. Test Compositions

The ovules are prepared by using Ovucire® (Ref 3460-Gattefosse, France)as the starting material. Ovucire® material is a blend of semi-syntheticglycerides comprising a mixture of saturated C₁₂-C₁₈ triglyceride fattyacids and additives. More precisely, this starting material is a mixtureof hard fat EP/NF/JPE (and) glyceryl ricinoleate (and) ethoxylated fattyalcohols (ceteth-20, steareth-20) EP/NF The melting point of Ovucire® isin the range of 32.5° C.-34.0° C. Ovucire® starting material is underthe form of waxy solid pellets at temperature of the laboratory.

The ovules are prepared by using a conventional technique. Ovules arestored refrigerated until the time of their usage.

D. Experimentation Schedule

8-10 weeks-old female Sprague Dawley rats (Janvier, France) are firstacclimated during a period of time of one week or more.

The acclimated rats undergo a bilateral ovariectomy (at “Day 0”), so asto induce an experimental menopause.

Three weeks after ovariectomy (at Day 21), the ovariectomized rats aredivided in five distinct groups of at least eight animals per group,respectively:

-   -   Group 1: the ovariectomized rats receive no treatment at all    -   Group 2: the ovariectomized rats are administered once daily        with an ovule comprising a liquid saline (placebo)    -   Group 3: the ovariectomized rats are administered once daily        with an ovule comprising estradiol alone, in the absence of        vitamin D analog    -   Group 4: the ovariectomized rats are administered once daily        with an ovule comprising vitamin D analog alone, in the absence        of estradiol.    -   Group 5: the ovariectomized rats are administered once daily        with an ovule comprising (i) the combination of estradiol and        vitamin D analog.

Each of the Groups 1, 2, 3, 4 and 5 of ovariectomized rats receives theindicated treatment during a period of time of six weeks (thus until Day63).

The body weight of each tested animal is monitored twice a week.

Vaginal pH of each tested animal is monitored the day before the firsttreatment, and in all cases the day following the last treatment (at Day64).

Smears of vaginal tissue are collected from each of the tested animal bygentle scraping along the wall of the vagina the day before the firsttreatment and the day following the last treatment for determining theVaginal Maturation Index (VMI).

At the end of the experiment (at Day 64), animals are sacrificed and thevagina and uterus are collected and their respective wet weight isdetermined.

One part of the vagina tissue material is paraffin-embedded. Slices ofthe paraffin-embedded vaginal tissue are stained for studying andmeasuring the vaginal epithelium thickness as well as the vaginalepithelium morphology.

One part of the vagina tissue material is rapidly stored at −80° C. forfurther experiment.

E. Doses

The median local estradiol dose has been calculated from the minimalmarketed daily dose effective in women, namely 7.5 μg. Dosing wascalculated utilizing weight-based comparisons with human dosage aspreviously published in similar studies (Montoya T I et al., 2015; BiolReprod.;92(2):43). The dose in a 70 kg women delivers 7.5 μg. By weightcomparison, this translates into 0.11 μg/kg, and consequently into a0.043 μg estradiol dose in a 400 g animal.

The minimum estradiol dose has been calculated by the same method from 2μg in women; by weight comparison, this translates into 0.01 μgestradiol dose in a 400 g animal.

According to animal response to treatment a 10-fold higher dose than themedian dose could be the maximum estradiol dose. This translates into0.4 μg in a 400 g animal.

Dose of estradiol tested in animals is selected in the range of 0.01 μgto 0.4 μg estradiol dose in a 400 g animal.

The dose of calcitriol has been calculated by the same method. The doseof calcitriol chosen in women is 0.075 μg to 1.00 μg. By weightcomparison, this translates into 0.0004 μg to 0.006 μg.

E. Measures

The following parameters are used for characterizing the status of thevaginal epithelium in each of the groups 1 to 5 of animals tested:

-   -   vaginal epithelium thickness,    -   vaginal pH value before and after chronic treatment.    -   vaginal tissue wet weight after chronic treatment.    -   vaginal maturation index (VMI), which measurement is performed        on vaginal smears before and after chronic treatment.

E.1. Measures Performed on the Vaginal and Uterine Tissues

The day following the last treatment, the rats are anesthetized with anoverdose of urethane to:

-   -   measure vaginal pH using a pH-indicator strip (3 readings/rot)    -   collect a vaginal smear for VMI assessment (2 samples/rat).    -   harvest vaginal/uterine horns.

After cleaning the vaginal and uterus tissues:

-   -   vaginal tissue is weighed to obtain wet weight as an index of        tissue atrophy.    -   uterine horns are weighed to rule out (or not) a systemic effect        of the local treatment, either by placebo, estradiol, vitamin D        or the combination of estradiol and vitamin D.

Vaginal tissue is then separated into two parts:

-   -   one half is fixed in 10% neutral buffered formalin and        paraffin-embedded for epithelium thickness evaluation and        morphology (H&E coloration)    -   the second half will be immediately frozen and stored at −80° C.        for further evaluation.

E.2. Vaginal Maturation Index (VMI) Assessment

For each rat, 2 samples are taken by gentle scraping along the wall ofthe vagina.

Then, each sample is smeared on one slide and fixed using a specialcytology fixative (labofix-VWR ref 13356770).

Papanicolaou stain (RAL diagnostics) is performed to assess a VMI.

VMI is assessed as a ratio obtained through performing a random count of3 major cell types. VMI=% leucocytes, % nucleated cells, % cornifiedcells.

Predominance of leukocytes indicates vaginal atrophy (absence ofestrogen stimulation) and predominance of nucleated and cornified cellsindicates effectiveness of supplementation: Nucleated cells compriseparabasal cells and intermediate cells. Cornified cells consist of thesuperficial cells.

Cell counts are performed by digital slide scanning and image analysis,using an Aperio® AT2 apparatus marketed by the company Leica Biosystems(Germany), according to the following protocol:

analyzing images of 5 sections per slide (×40) will be used foranalysis. Thus, for each rat, a total of 10 sections will be reviewedfor VMI assessment.

E.3. Epithelium Thickness Evaluation

Vaginal tissue is embedded in paraffin, sectioned and stained withhematoxylin/eosine (2 slides/rat) using a fully automated multi-stainersystem for histological analysis, the Multistainer ST5020 in conjunctionwith the CV5030 Coverslipper marketed by the Company Leica Biosystems(Germany).

Digital slide scanning and image analysis for Vaginal epithelialthickness evaluation is performed according to the following protocol:

3 sections/slide: the first 2 sections (5 fields/section) of each slideis used for analysis. The 3^(rd) section is considered as a back-upsection. Thus, for each rat, a total of 20 fields is reviewed forhisto-morphometry.

F. Comments on the Experimental Results

Overall, the rats reacted well to the various treatment conditionstested, as it was confirmed by the regular body weight increase. As itis shown in FIG. 1 , treatment conditions with estradiol, calcitriol orcombination of estradiol and calcitriol, irrespective of the dose ofestradiol or calcitriol that were used, did not cause any change in theanimal body weight. The results depicted in FIG. 1 showed that nosignificant difference in body weight evolution among the differentexperimental groups was observed at the end of the treatment timeperiod.

Further, the results showed no difference in the vaginal pH value of theanimals at the start of the experiment, nor a vaginal pH valuedifference at the end of the treatment time period. This is probably dueto the low sensitivity of pH-indicator strips to detect smallvariability or to the large interindividual variability.

Epithelium thickness was measured as it directly illustrates the effectof a given treatment on vaginal atrophy. As shown in FIG. 2 , a 0.4 μgdose of estradiol induces a high increase in the vaginal epitheliumthickness, as compared to placebo (the dashed line in FIG. 2 ).Calcitriol alone, irrespective of the dose which has been tested, didnot cause any change in the vaginal epithelium thickness. However, whena low dose of estradiol was combined to a non-active dose of calcitriol,a substantial increase in the vaginal epithelium thickness was observed.FIG. 2 shows that the combination of low combination doses such as 0.02μg estradiol with 0.006 μg calcitriol cause an increase in the vaginalepithelium thickness that is of the same order than a twenty fold higherdose of estradiol alone. At the dose of 0.02 μg estradiol, that isadministered locally, there is a reduced risk of systemic passage ofbiologically active amounts of estradiol. Especially, at the dose of0.02 μg of local administration of estradiol, systemic adverse effectsare avoided or at least considerably reduced. Almost the same remarksmay be made regarding the 0.2 μg estradiol combination treatmentconditions, although the risk of systemic activity may be increased.

Regarding the assessment of the Vaginal Maturation Index (VMI), beyondthe vaginal epithelium thickness, an important parameter resides in thecellular constitution of the vaginal epithelium, which includes thecontent of the vaginal epithelium in cornified cells and leukocytes,respectively.

As regards the vaginal epithelium cornified cells layer, FIG. 3Aillustrates a histological view of the vaginal epithelium of anovariectomized rat having received locally placebo-containing ovulesonly. FIG. 3A shows the stratum germinativum which is composed ofstratum basale as a single layer of columnar cells and outer stratumspinosum (the darker external cell layer in FIG. 3A) as multiple layersof polyhedral cells. The cornified cells layer locates us the thin anddiscontinuous extreme outer cell layer in FIG. 3A. The histologicalphotograph of FIG. 3B shows that a 0.02 μg dose of estradiol does notcause a detectable change in the cornified cell layer, whereas thehistological photographs of FIGS. 3C and 3D illustrate that thecornified cell layer have a thickness and a density which increase withthe increasing doses of 0.08 μg and 0.4 μg estradiol.

Further, the results depicted in FIG. 3 show that although calcitriolinduced no significant change in the content of the vaginal epitheliumcornified cells, irrespectively of the doses which were tested, even thecombination of the lowest doses of estradiol and calcitriol,respectively, causes a high increase in the cornified cells content,which denotes a highly positive effect on the maturation of the vaginalepithelium, i.e. a recovery of the mature vaginal epithelium structure,and thus a recovery from the experimentally-induced vaginal atrophy.

As shown in FIG. 5 , the combination of very low doses of 0.02 μgestradiol and 0.006 μg calcitriol, thus at doses wherein calcitriolalone causes no effect, causes a high decrease in the vaginal epitheliumcontent in leukocytes, which is another result confirming that thecombination treatments tested reversed the experimentally-inducedvaginal atrophy.

Also, as shown in FIG. 6 , the calcium plasma level was significantlyincreased at the doses of 0.08 μg and 0.1 μg, respectively.

However, it may be noticed that the use of calcitriol dose as low as0.006 μg did not cause any significant change in the calcium plasmalevel.

Altogether, the results presented in this example show the effectivenessof the combination of estradiol and calcitriol at low doses for treatingvaginal atrophy.

It may be added that at certain low dose ranges of the combinedtreatment, the said combined treatment may allow substantially avoidingundesirable side effects, such as (i) significant systemic presence ofestradiol and (ii) plasma calcium level increase.

The invention claimed is:
 1. A vaginal composition comprising, as activeingredients, a combination consisting of estrogen and vitamin D or avitamin D analog at a daily dosage delivery of (i) estrogen ranging from1 μg to 10 μg estrogen of estradiol equivalent and (ii) vitamin D oranalog ranging from 7.5 μg to 100 μg of vitamin D equivalent.
 2. Thevaginal composition according to claim 1, wherein the daily dosagedelivery of estrogen ranges from 1 μg to 7.5 μg of estradiol equivalent.3. The vaginal composition according to claim 1 wherein the saidcomposition has a liquid, solid or semi-solid presentation.
 4. Thevaginal composition according to claim 1 wherein said vaginalcomposition is a cream or a gel composition.
 5. The vaginal compositionaccording to claim 1 presented as daily unit dosage form of asuppository.
 6. The vaginal composition according to claim 5, whereinthe suppository is selected from a group consisting of a capsule, anovule and a tablet.
 7. The vaginal composition according to claim 1which is comprised in a delivery device selected from the groupconsisting of a transmucosal device and a vaginal ring.
 8. The vaginalcomposition according to claim 1 wherein the said estrogen is selectedfrom the group consisting of estradiol, estradiol valerate, estradiolbenzoate, estradiol 17β-cypionate, estradiol dipropionate, estradiolenanthate, estropipate, equilenin, equilin, estriol, estriol succinate,estrone, ethinyl estradiol, estetrol, quinestrol, quinestranol,conjugated estrogens, esterified estrogens, and mixtures thereof.
 9. Thevaginal composition according to claim 1 wherein the said estrogen isestradiol or a derivative thereof.
 10. The vaginal composition accordingto claim 1 wherein the vitamin D analog is selected from the groupconsisting of cholecalciferol, ergocalciferol, paricalcitol,doxercalciferol, dihydrotachysterol and derivatives thereof.
 11. Thevaginal composition according to claim 1, wherein the vitamin D analogis selected from the group consisting of calcitriol and calcipotriol.12. A method for treating vaginal atrophy in a subject, comprisingproviding to a vagina of the subject as active ingredients a combinationconsisting of estrogen and vitamin D or vitamin D analog at a dailydosage delivery of (i) estrogen ranging from 1 μg to 10 μg estrogen ofestradiol equivalent and (ii) vitamin D or analog ranging from 7.5 μg to100 μg of vitamin D equivalent.
 13. The method of claim 12, wherein thedaily dosage delivery of estrogen ranges from 1 μg to 7.5 μg ofestradiol equivalent.
 14. A method for treating vaginal atrophy in asubject, comprising providing to a vagina of the subject as activeingredients a combination consisting of estradiol and calcitriol at adaily dosage delivery of (i) estradiol ranging from 1 μg to 10 μg and(ii) calcitriol ranging from 0.075 μg to 1 μg.
 15. The vaginalcomposition according to claim 1, wherein the daily dosage delivery ofestrogen ranges from 1 μg to 7.5 μg of estradiol equivalent and saidcomposition is a vaginal tablet.
 16. The method according to claim 12,wherein the daily dosage delivery of estrogen ranges from 1 μg to 7.5 μgof estradiol equivalent and said composition is a vaginal tablet. 17.The method according to claim 14, wherein the daily dosage delivery ofestradiol ranges from 1 μg to 7.5 μg and said composition is a vaginaltablet.
 18. The method of claim 16, wherein the daily dosage delivery ofestrogen ranges from 1 μg to 7.5 μg of estradiol equivalent.